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WO2024255790A1 - Composés cycliques fusionnés, composition pharmaceutique le comprenant et leur utilisation - Google Patents

Composés cycliques fusionnés, composition pharmaceutique le comprenant et leur utilisation Download PDF

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Publication number
WO2024255790A1
WO2024255790A1 PCT/CN2024/098964 CN2024098964W WO2024255790A1 WO 2024255790 A1 WO2024255790 A1 WO 2024255790A1 CN 2024098964 W CN2024098964 W CN 2024098964W WO 2024255790 A1 WO2024255790 A1 WO 2024255790A1
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Prior art keywords
compound
ring
alkyl
membered
synthesis
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Chinese (zh)
Inventor
张贵平
李家鹏
王奎锋
郑计岳
刘涛
徐浩杰
姬飞虹
童水龙
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Suzhou Genhouse Bio Co Ltd
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Suzhou Genhouse Bio Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems

Definitions

  • the present invention relates to a condensed ring compound, a pharmaceutical composition containing the condensed ring compound, and use thereof for preventing or treating diseases.
  • Microsatellite instability is a type of genomic damage caused by mismatch repair deficiency (dMMR).
  • dMMR mismatch repair deficiency
  • MSI-H microsatellite high instability
  • WRN Werner helicase
  • MSI-H cancers see, e.g., Chan EM et al., Nature. 2019 Apr; 568(7753): 551-556.
  • WRN is a member of the RecQ family of DNA helicases and plays an important role in maintaining genome stability, DNA repair, replication, transcription, and telomere maintenance.
  • Studies on WRN-dependent mechanisms have shown that dinucleotide TA repeats are massively amplified in MSI cells. These amplified TA repeats form secondary DNA structures that require WRN helicases to unwind (see, e.g., van Wietmarschen N et al., Nature. 2020 Oct; 586(7828): 292-298).
  • WRN WRN helicases
  • MSI-H microsatellite instability-high
  • dMMR mismatch repair deficiency
  • the present application provides compounds used as WRN inhibitors, which can be used to prevent or treat cancers characterized by microsatellite high instability (MSI-H) or mismatch repair deficiency (dMMR).
  • the compounds of the present invention also have excellent properties such as good physicochemical properties (such as solubility, physical and/or chemical stability), good pharmacokinetic properties (such as improved bioavailability, good metabolic stability, suitable half-life and duration of action), good safety (lower toxicity (such as reduced cardiotoxicity) and/or less side effects), less prone to drug resistance.
  • One aspect of the present invention provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, atropisomer, tautomer, polymorph, solvate, metabolite, isotopically labeled compound or prodrug thereof, wherein the compound has a structure of formula (I):
  • R 1 is at the position marked with "*” in the above group connected
  • R 2 is connected to the position marked with "**” in the above group
  • R 3 is connected to the position marked with "***” in the above group
  • R 4 is connected to the position marked with "****" in the above group
  • R 1 is selected from -NR 11 R 12 ,
  • R 3 , R 11 , R 12 , R 13 , R 21 , R 22 , R 23 and R 24 are each independently selected at each occurrence from H, halogen, -OH, -NH 2 , -CN, -NO 2 , -SF 5 , C 1-6 alkyl, deuterated C 1-6 alkyl, halogenated C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, 5-14 membered heteroaryl, C 6-12 aralkyl, -C( ⁇ O)R a , -OC( ⁇ O)R a , -C( ⁇ O)OR a , -OR a , -SR a , -S( ⁇ O)R a , -S( ⁇ O) 2 R a , -S( ⁇ O) 2 NR a R b -NRa
  • R 14 at each occurrence is independently selected from -L 1 -(C 3-6 cycloalkyl), -L 1 -(3-10 membered heterocyclyl), -L 1 -(C 6-10 aryl) and -L 1 -(5-14 membered heteroaryl);
  • R 41 is selected from a C 3-6 hydrocarbon ring, a 3-10 membered heterocyclic ring, a C 6-10 aromatic ring, and a 5-14 membered heteroaromatic ring;
  • Ring C, Ring D, Ring E, Ring X and Ring Z are each independently selected from a C 3-6 hydrocarbon ring, a 3-10 membered heterocyclic ring, a C 6-10 aromatic ring and a 5-14 membered heteroaromatic ring;
  • Ring Y is absent or is selected from a C 3-6 hydrocarbon ring, a 3-10 membered heterocyclic ring, a C 6-10 aromatic ring and a 5-14 membered heteroaromatic ring; when Ring Y is absent, R 24 is also absent;
  • R, Ra and Rb are each independently selected at each occurrence from H, C1-6 alkyl, C3-10 cycloalkyl, 3-10 membered heterocyclyl, C6-10 aryl, 5-14 membered heteroaryl and C6-12 aralkyl;
  • n, p and q are each independently an integer selected from 1, 2 or 3;
  • n is an integer of 0 or 1.
  • Another aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a prophylactically or therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, metabolite, isotopically labeled compound or prodrug thereof and one or more pharmaceutically acceptable carriers.
  • Another aspect of the present invention provides use of a compound of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, metabolite, isotope-labeled compound or prodrug thereof, or a pharmaceutical composition of the present invention in the preparation of a medicament for use as a WRN inhibitor.
  • Another aspect of the present invention provides a method for preventing or treating cancer (preferably a cancer characterized by microsatellite high instability (MSI-H) or mismatch repair deficiency (dMMR)), which comprises administering to an individual in need thereof an effective amount of a compound of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, metabolite, isotope-labeled compound or prodrug thereof, or a pharmaceutical composition of the present invention.
  • cancer preferably a cancer characterized by microsatellite high instability (MSI-H) or mismatch repair deficiency (dMMR)
  • Zwitterion or "zwitterionic form” means a compound containing both positively charged and negatively charged functional groups.
  • certain groups are in non-zwitterionic form per se, but in a tautomer in which a proton is shifted, the group is in zwitterionic form.
  • R 41 group in the compound described herein is a pyridine ring substituted with at least one -OH, it may be in the following zwitterionic form (c) or non-zwitterionic form (d) or a mixture of the two forms:
  • R 100 , R 101 , and R 102 represent substituents as defined herein;
  • R 41 group in the compound described herein when it is a pyrimidine ring substituted with at least one -OH, it may be in the following zwitterionic form (a) or (b) or non-zwitterionic form (e), or a mixture of any two or three of the forms:
  • R 100 and R 102 represent substituents as defined herein.
  • alkylene refers to a saturated divalent hydrocarbon group, preferably a saturated divalent hydrocarbon group having 1, 2, 3, 4, 5 or 6 carbon atoms, such as methylene, ethylene, propylene or butylene.
  • alkyl is defined as a straight or branched saturated aliphatic hydrocarbon. In some embodiments, the alkyl has 1 to 12, for example 1 to 6 carbon atoms.
  • C 1-6 alkyl refers to a linear or branched group of 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl or n-hexyl), which is optionally substituted by 1 or more (such as 1 to 3) suitable substituents such as halogen (in this case, the group is referred to as "haloalkyl”) (e.g., CF 3 , C 2 F 5 , CHF 2 , CH 2 F, CH 2 CF 3 , CH 2 Cl or -CH 2 CH 2 CF 3, etc.).
  • haloalkyl e.g., CF 3
  • C 1-4 alkyl refers to a linear or branched aliphatic hydrocarbon chain of 1 to 4 carbon atoms (ie, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl).
  • alkenyl means a linear or branched monovalent hydrocarbon radical containing one or more double bonds and having 2 to 6 carbon atoms (“ C2-6 alkenyl”).
  • alkynyl refers to a monovalent hydrocarbon group containing one or more triple bonds, preferably having 2, 3, 4, 5 or 6 carbon atoms, such as ethynyl, 2-propynyl, 2-butynyl, 1,3-butadiynyl, etc.
  • the alkynyl group is optionally substituted with one or more (such as 1 to 3) identical or different substituents.
  • alkynylene is a corresponding divalent group, including, for example, “C 2-8 alkynylene", “C 2-6 alkynylene", “C 2-4 alkynylene", etc. Examples thereof include, but are not limited to The alkynylene group is optionally substituted with one or more (such as 1 to 3) identical or different substituents.
  • paracyclic ring or “fused ring” refers to a ring system formed by two or more cyclic structures sharing two adjacent atoms with each other.
  • spirocycle refers to a ring system formed by two or more cyclic structures that share one ring atom with each other.
  • bridged ring refers to a ring system formed by two or more cyclic structures sharing two atoms that are not directly connected to each other.
  • cycloalkylene refers to saturated (i.e., “cycloalkylene” and “cycloalkyl”) or partially unsaturated (i.e., having one or more double bonds and/or triple bonds within the ring) monocyclic or polycyclic hydrocarbon rings (including spiro, fused or bridged ring systems) having, for example, 3-10 (suitably 3-8, more suitably 3-6) ring carbon atoms, including but not limited to (cyclo)propyl (ring), (cyclo)butyl (ring), (cyclo)pentyl (ring), (cyclo)hexyl (ring), (cyclo)heptyl (ring), (cyclo)octyl (ring), (cyclo)nonyl (ring), (cyclo)hexenyl (ring) and the like.
  • the cycloalkyl group has 3 to 15 carbon atoms.
  • C3-6cycloalkyl refers to a saturated monocyclic or polycyclic (such as bicyclic) hydrocarbon ring of 3 to 6 ring carbon atoms (e.g., cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl), which is optionally substituted with 1 or more (such as 1 to 3) suitable substituents, for example, methyl-substituted cyclopropyl.
  • the heterocyclyl may be attached to the rest of the molecule via the carbon atoms and/or heteroatoms (if present).
  • a 3-10 membered heterocyclyl group is a group having 3-10 carbon atoms and heteroatoms in the ring, such as, but not limited to, an oxirane, an aziridinyl, an azetidinyl, an oxetanyl, a tetrahydrofuranyl, a dioxolinyl, a pyrrolidinyl, a pyrrolidonyl, an imidazolidinyl, a pyrazolidinyl, a pyrrolinyl, a tetrahydropyranyl, a piperidinyl, a morpholinyl, a dithianyl, a thiomorpholinyl, a piperazinyl or a trithianyl.
  • heterocyclyl encompasses fused ring structures, where the point of attachment to other groups can be on either ring of the fused ring structure. Therefore, the heterocyclic group of the present invention also includes, but is not limited to, heterocyclic and heterocyclic groups, heterocyclic and cycloalkyl groups, monoheterocyclic and monoheterocyclic groups, monoheterocyclic and monocycloalkyl groups, aryl and heterocyclic groups, heteroaryl and heterocyclic groups, such as 3-7 membered (mono) heterocyclic groups and 3-7 membered (mono) heterocyclic groups, 3-7 membered (mono) heterocyclic groups and (mono) cycloalkyl groups, 3-7 membered (mono) heterocyclic groups and C 4-6 (mono) cycloalkyl groups, C 6-10 aryl and 3-7 membered heterocyclic groups, 5-6 membered heteroaryl and 3
  • heterocyclyl encompasses bridged heterocyclyls (bridged heterocycle) and spiro heterocyclyls (spiro heterocycle).
  • bridged heterocycle refers to a cyclic structure containing one or more (e.g., 1, 2, 3 or 4) heteroatoms (e.g., oxygen atoms, nitrogen atoms and/or sulfur atoms) formed by two rings sharing two ring atoms that are not directly connected, including but not limited to 7-10 membered bridged heterocycles, 8-10 membered bridged heterocycles, 7-10 membered nitrogen-containing bridged heterocycles, 7-10 membered oxygen-containing bridged heterocycles, 7-10 membered sulfur-containing bridged heterocycles, etc., for example
  • the "nitrogen-containing bridged heterocycle", “oxygen-containing bridged heterocycle” and “sulfur-containing bridged heterocycle” optionally further contain one or more other heteroatoms selected from oxygen, nitrogen and sulfur.
  • spiroheterocycle refers to a cyclic structure containing one or more (e.g., 1, 2, 3, or 4) heteroatoms (e.g., oxygen atoms, nitrogen atoms, sulfur atoms) formed by two or more rings sharing a ring atom, including but not limited to 5-10 membered spiroheterocycle, 6-10 membered spiroheterocycle, 6-10 membered nitrogen-containing spiroheterocycle, 6-10 membered oxygen-containing spiroheterocycle, 6-10 membered sulfur-containing spiroheterocycle, etc., for example
  • the "nitrogen-containing spiro heterocycle", “oxygen-containing spiro heterocycle” and “sulfur-containing spiro heterocycle” optionally further contain one or more other heteroatoms selected from oxygen, nitrogen and sulfur.
  • 6-10 membered nitrogen-containing spiro heterocyclic group refers to a spiro heterocyclic group containing a total of 6-10 ring atoms and at least one of the ring atoms being a nitrogen atom.
  • (ylidene)aryl and “aromatic ring” refer to an all-carbon monocyclic or fused-ring polycyclic aromatic group having a conjugated ⁇ electron system.
  • C 6-10 (ylidene)aryl and “C 6-10 aromatic ring” mean an aromatic group containing 6 to 10 carbon atoms, such as (ylidene)phenyl (benzene ring) or (ylidene)naphthyl (naphthalene ring).
  • the (ylidene)aryl group and the aromatic ring are optionally substituted with 1 or more (such as 1 to 3) suitable substituents (e.g., halogen, -OH, -CN, -NO 2 , C 1-6 alkyl, etc.).
  • suitable substituents e.g., halogen, -OH, -CN, -NO 2 , C 1-6 alkyl, etc.
  • aralkyl refers to an alkyl substituted with an aryl group, wherein the aryl group and the alkyl group are as defined herein. Typically, the aryl group may have 6-14 carbon atoms, and the alkyl group may have 1-6 carbon atoms. Exemplary aralkyl groups include, but are not limited to, benzyl, phenylethyl, phenylpropyl, phenylbutyl.
  • heteroaryl(ene) and “heteroaromatic ring” refer to a monocyclic, bicyclic or tricyclic aromatic ring system having 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 ring atoms, in particular 1 or 2 or 3 or 4 or 5 or 6 or 9 or 10 carbon atoms, and which contains at least one heteroatom which may be identical or different (the heteroatom being, for example, oxygen, nitrogen or sulfur) and, in each case additionally may be benzo-fused.
  • (ene)heteroaryl or “heteroaryl ring” is selected from (ene)thienyl (ring), (ene)furanyl (ring), (ene)pyrrolyl (ring), (ene)oxazolyl (ring), (ene)thiazolyl (ring), (ene)imidazolyl (ring), (ene)pyrazolyl (ring), (ene)isoxazolyl (ring), (ene)isothiazolyl (ring), (ene)oxadiazolyl (ring), (ene)triazolyl (ring), (ene)thiadiazolyl (ring), etc., and their benzo derivatives; or (ene)pyridinyl (ring), (ene)pyridazinyl (ring), (ene)pyrimidinyl (ring), (ene)pyrazinyl (ring), (ene)triazinyl (ring), etc., and their benzo derivatives.
  • halo or halogen group is defined to include F, Cl, Br, or I.
  • alkylthio refers to an alkyl group as defined above attached to the parent molecular moiety through a sulfur atom.
  • Representative examples of C 1-6 alkylthio include, but are not limited to, methylthio, ethylthio, tert-butylthio and hexylthio.
  • the nitrogen-containing heterocycle is preferably a saturated nitrogen-containing monocyclic ring.
  • the 3- to 14-membered nitrogen-containing heterocycle is A group having 3-14 carbon atoms and heteroatoms (at least one of which is a nitrogen atom) in the ring, including but not limited to a three-membered nitrogen-containing heterocycle (such as aziridine), a four-membered nitrogen-containing heterocycle (such as azetidinyl), a five-membered nitrogen-containing heterocycle (such as pyrrolyl, pyrrolidinyl (pyrrolidine ring), pyrrolinyl, pyrrolidonyl, imidazolyl, imidazolidinyl, imidazolinyl, pyrazolyl, pyrazolinyl), a six-membered nitrogen-containing heterocycle (such as piperidinyl (piperidine ring), morpholinyl, thiomorpholinyl, piperazinyl), a seven-membered nitrogen-containing heterocycle, etc.
  • substituted means that one or more (e.g., one, two, three, or four) hydrogens on the designated atom are replaced by a selection from the indicated group, provided that the normal valence of the designated atom in the present context is not exceeded and the substitution forms a stable compound. Combinations of substituents and/or variables are permitted only if such combinations form stable compounds.
  • substituent may be (1) unsubstituted or (2) substituted. If a carbon of a substituent is described as being optionally substituted with one or more of the listed substituents, then one or more hydrogens on the carbon (to the extent of any hydrogens present) may be replaced, individually and/or together, with independently selected optional substituents. If a nitrogen of a substituent is described as being optionally substituted with one or more of the listed substituents, then one or more hydrogens on the nitrogen (to the extent of any hydrogens present) may each be replaced with an independently selected optional substituent.
  • each substituent is selected independently of the other.
  • each substituent may be the same as or different from another (other) substituent.
  • one or more means 1 or more than 1, such as 2, 3, 4, 5 or 10, where reasonable.
  • the point of attachment of a substituent may be from any suitable position of the substituent.
  • the present invention also includes all pharmaceutically acceptable isotopically labeled compounds which are identical to the compounds of the present invention except that one or more atoms are replaced by an atom having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number prevalent in nature.
  • isotopes suitable for inclusion in the compounds of the present invention include, but are not limited to, isotopes of hydrogen (e.g., deuterium (D, 2 H), tritium (T, 3 H)); isotopes of carbon (e.g., 11 C, 13 C, and 14 C); isotopes of chlorine (e.g., 36 Cl); isotopes of fluorine (e.g., 18 F); isotopes of iodine (e.g., 123 I and 125 I); isotopes of nitrogen (e.g., 13 N and 15 N); isotopes of oxygen (e.g., 15 O, 17 O, and 18 O); isotopes of phosphorus (e.g., 32 P); and isotopes of sulfur (e.g., 35 S).
  • isotopes of hydrogen e.g., deuterium (D, 2 H), tritium (T, 3 H)
  • Certain isotopically labeled compounds of the invention are useful in drug and/or substrate tissue distribution studies (e.g., assays).
  • the radioisotopes tritium (i.e., 3 H) and carbon-14 (i.e., 14 C) are particularly useful for this purpose because they are easily incorporated and easily detected.
  • Substitution with positron emitting isotopes e.g., 11 C, 18 F, 15 O, and 13 N
  • PET positron emission tomography
  • Isotopically labeled compounds of the invention can be prepared by methods similar to those described in the accompanying routes and/or in the examples and preparations by using appropriate isotopically labeled reagents in place of the non-labeled reagents previously employed.
  • Pharmaceutically acceptable solvates of the invention include those in which the crystallization solvent may be isotopically substituted, for example, D 2 O, acetone-d 6 or DMSO-d 6 .
  • stereoisomer means an isomer formed due to at least one asymmetric center. In compounds with one or more (e.g., one, two, three, or four) asymmetric centers, it can produce racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers. Specific individual molecules can also exist as geometric isomers (cis/trans). Similarly, the compounds of the present invention can exist as mixtures (commonly referred to as tautomers) of two or more structurally different forms in rapid equilibrium. Representative examples of tautomers include keto-enol tautomers, phenol-ketone tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc.
  • solid lines can be used Solid wedge Virtual wedge Carbon-carbon bonds of the compounds of the invention.
  • the use of solid lines to depict bonds to asymmetric carbon atoms is intended to indicate that all possible stereoisomers at that carbon atom are included (e.g., specific enantiomers, racemic mixtures, etc.).
  • the use of solid or dashed wedges to depict bonds to asymmetric carbon atoms is intended to indicate that the stereoisomer shown is present. When present in a racemic mixture, the solid and dashed wedges are used to define relative stereochemistry, not absolute stereochemistry.
  • the compounds of the invention are intended to exist in the form of stereoisomers, which include cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, atropisomers, and mixtures thereof.
  • the compounds of the invention may exhibit more than one type of isomerism and consist of mixtures thereof (e.g., racemic mixtures and diastereomeric pairs).
  • Atropisomers are compounds that can be separated into their rotationally restricted isomers.
  • compositions of the present invention may be present in free form for treatment, or, where appropriate, in the form of pharmaceutically acceptable derivatives thereof.
  • pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, esters, solvates, metabolites or prodrugs, which, upon administration to a patient in need thereof, are capable of directly or indirectly providing the compounds of the present invention. or its metabolites or residues. Therefore, when referring to "compounds of the present invention" herein, the above-mentioned various derivative forms of the compounds are also intended to be encompassed.
  • Pharmaceutically acceptable salts of the compounds of the present invention include acid addition salts and base addition salts thereof.
  • esters means an ester derived from the compounds of the general formulae herein, including physiologically hydrolyzable esters (which can be hydrolyzed under physiological conditions to release the compounds of the present invention in free acid or alcohol form).
  • physiologically hydrolyzable esters which can be hydrolyzed under physiological conditions to release the compounds of the present invention in free acid or alcohol form.
  • the compounds of the present invention themselves may also be esters.
  • the compounds of the present invention may exist in the form of solvates (preferably hydrates), wherein the compounds of the present invention contain polar solvents as structural elements of the crystal lattice of the compounds, in particular water, methanol or ethanol.
  • polar solvents as structural elements of the crystal lattice of the compounds, in particular water, methanol or ethanol.
  • the amount of polar solvents, in particular water, may be present in a stoichiometric or non-stoichiometric ratio.
  • metabolites of the compounds of the present invention i.e., substances formed in vivo upon administration of the compounds of the present invention. Such products may be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic hydrolysis, etc. of the administered compound.
  • the present invention includes metabolites of the compounds of the present invention, including compounds prepared by contacting the compounds of the present invention with a mammal for a period of time sufficient to produce a metabolic product thereof.
  • the present invention further includes within its scope prodrugs of the compounds of the present invention, which are certain derivatives of the compounds of the present invention that may themselves have little or no pharmacological activity and can be converted into compounds of the present invention having the desired activity when administered into or onto the body, for example, by hydrolytic cleavage.
  • prodrugs will be functional group derivatives of the compounds that are easily converted into the desired therapeutically active compounds in vivo. Additional information on the use of prodrugs can be found in "Pro-drugs as Novel Delivery Systems," Vol. 14, ACS Symposium Series (T. Higuchi and V. Stella) and "Bioreversible Carriers in Drug Design," Pergamon Press, 1987 (E. B. Roche, ed., American Pharmaceutical Association).
  • Prodrugs of the present invention can be prepared, for example, by replacing appropriate functional groups present in the compounds of the present invention with certain moieties known to those skilled in the art as "pro-moieties” (e.g. as described in “Design of Prodrugs", H. Bundgaard (Elsevier, 1985)).
  • the present invention also encompasses compounds of the present invention containing protecting groups.
  • protecting groups In any process for preparing the compounds of the present invention, it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules involved, thereby forming a chemically protected form of the compounds of the present invention. This can be achieved by conventional protecting groups, for example, those described in Protective Groups in Organic Chemistry, ed. J.F.W. McOmie, Plenum Press, 1973; and T.W. Greene & P.G.M. Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991, which references are incorporated herein by reference.
  • the protecting groups may be removed at an appropriate subsequent stage using methods known in the art.
  • the term "about” means within ⁇ 10% of the stated numerical value, preferably within ⁇ 5%, and more preferably within ⁇ 2%.
  • the present disclosure provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, atropisomer, tautomer, polymorph, solvate, metabolite, isotopically labeled compound, or prodrug thereof, wherein the compound has the structure of Formula (I):
  • R 1 is connected to the position marked with “*” in the above group
  • R 2 is connected to the position marked with “**” in the above group
  • R 3 is connected to the position marked with “***” in the above group
  • R 4 is connected to the position marked with “****” in the above group
  • R 1 is selected from -NR 11 R 12 ,
  • R 3 , R 11 , R 12 , R 13 , R 21 , R 22 , R 23 and R 24 are each independently selected at each occurrence from H, halogen, -OH, -NH 2 , -CN, -NO 2 , -SF 5 , C 1-6 alkyl, deuterated C 1-6 alkyl, halogenated C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, 5-14 membered heteroaryl, C 6-12 aralkyl, -C( ⁇ O)R a , -OC( ⁇ O)R a , -C( ⁇ O)OR a , -OR a , -SR a , -S( ⁇ O)R a , -S( ⁇ O) 2 R a , -S( ⁇ O) 2 NR a R b -NRa
  • R 14 at each occurrence is independently selected from -L 1 -(C 3-6 cycloalkyl), -L 1 -(3-10 membered heterocyclyl), -L 1 -(C 6-10 aryl) and -L 1 -(5-14 membered heteroaryl);
  • R 41 is selected from a C 3-6 hydrocarbon ring, a 3-10 membered heterocyclic ring, a C 6-10 aromatic ring, and a 5-14 membered heteroaromatic ring;
  • Ring C, Ring D, Ring E, Ring X and Ring Z are each independently selected from a C 3-6 hydrocarbon ring, a 3-10 membered heterocyclic ring, a C 6-10 aromatic ring and a 5-14 membered heteroaromatic ring;
  • Ring Y is absent or is selected from a C 3-6 hydrocarbon ring, a 3-10 membered heterocyclic ring, a C 6-10 aromatic ring and a 5-14 membered heteroaromatic ring; when Ring Y is absent, R 24 is also absent;
  • R, Ra and Rb are each independently selected at each occurrence from H, C1-6 alkyl, C3-10 cycloalkyl, 3-10 membered heterocyclyl, C6-10 aryl, 5-14 membered heteroaryl and C6-12 aralkyl;
  • n, p and q are each independently an integer selected from 1, 2 or 3;
  • n is an integer of 0 or 1.
  • the present disclosure provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, atropisomer, tautomer, polymorph, solvate, metabolite, isotopically labeled compound or prodrug thereof, wherein the compound has a structure of the following formula:
  • R 13 is H, halogen or C 1-6 alkyl
  • R 14 is selected from 3-10 membered heterocyclyl, -(C 1-6 alkylene)-(3-10 membered heterocyclyl) and -O-(C 1-6 alkylene)-(3-10 membered heterocyclyl), said heterocyclyl being optionally substituted with one or more substituents independently selected from the group consisting of C 1-6 alkyl, C 3-6 cycloalkyl, 3-10 membered heterocyclyl, -C 1-6 alkylene-C 3-6 cycloalkyl and -C 1-6 alkylene-CN; and
  • n is an integer of 1 or 2.
  • R 13 is H, halogen or C 1-6 alkyl
  • R 14 is selected from 3-10 membered heterocyclyl, -(C 1-6 alkylene)-(3-10 membered heterocyclyl) and -O-(C 1-6 alkylene)-(3-10 membered heterocyclyl), wherein the heterocyclyl is optionally substituted with one or more substituents independently selected from the group consisting of C 1-6 alkyl, C 3-6 cycloalkyl, -C 1-6 alkylene-C 3-6 cycloalkyl and -C 1-6 alkylene-CN; and
  • n is an integer of 1 or 2.
  • R 1 is
  • R 1 is
  • Ring C is a benzene ring, a 5-6 membered heterocyclic ring or a 5-6 membered heteroaromatic ring;
  • Ring D is a benzene ring or a 5-6 membered heteroaromatic ring
  • the above-mentioned benzene ring, heterocycle and heteroaromatic ring are each optionally substituted by one or more substituents independently selected from the following: halogen, -OH, -NH2 , -CN, -NO2 , C1-6 alkyl, deuterated C1-6 alkyl, C1-6 haloalkyl, -OC1-6 alkyl, C3-6 cycloalkyl and 3-10 membered heterocyclyl; preferably, the above-mentioned benzene ring, heterocycle and heteroaromatic ring are each optionally substituted by one or more substituents independently selected from the following: halogen, C1-6 alkyl, C1-6 haloalkyl, -OC1-6 alkyl and C3-6 cycloalkyl.
  • R 1 is
  • R 1 is
  • R1 is -NR11R12 ;
  • R 11 and R 12 are each independently selected from H, C 1-6 alkyl, C 3-6 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, 5-14 membered heteroaryl, C 6-12 aralkyl, -C 1-6 alkylene-R a and -C 1-6 alkylene-NR a R b ;
  • Ra and Rb are each independently selected at each occurrence from H, C1-6 alkyl, C3-10 cycloalkyl, 3-10 membered heterocyclyl, C6-10 aryl, 5-14 membered heteroaryl and C6-12 aralkyl;
  • alkylene, alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl and aralkyl groups are each optionally substituted at each occurrence with one or more substituents independently selected from the group consisting of halogen and C 1-6 alkyl.
  • R 1 is
  • R 1 is
  • n is an integer of 1 or 2.
  • Ring E is a C 3-6 hydrocarbon ring, a 3-10 membered heterocyclic ring, a C 6-10 aromatic ring or a 5-14 membered heteroaromatic ring;
  • R 13 is H, halogen, C 1-6 alkyl, 3-10 membered heterocyclyl, -C( ⁇ O)-(C 1-6 alkyl), -O-(C 1-6 alkyl) or -N(C 1-6 alkyl) 2 , wherein the alkyl and heterocyclyl are further optionally substituted by C 1-6 alkyl or 3-10 membered heterocyclyl;
  • n is an integer of 1 or 2.
  • Ring E is a 3-10 membered heterocyclic ring, a C 6-10 aromatic ring or a 5-14 membered heteroaromatic ring;
  • R 13 is H, halogen, C 1-6 alkyl, -C( ⁇ O)-(C 1-6 alkyl) or -O-(C 1-6 alkyl);
  • n is an integer of 1 or 2.
  • R 1 is
  • R 1 is
  • R 1 is
  • Ring X is a 5-6 membered heterocyclic ring (eg, a pyridone ring) or a 5 membered heteroaromatic ring; or
  • Ring X is a benzene ring, and at least one R 23 is (—C 3-6 cycloalkylene)-CN or (—C 3-6 cycloalkylene)-C 1-6 haloalkyl.
  • Ring X is a benzene ring and Ring Y is a 5-6 membered heterocyclic ring or a 5-6 membered heteroaromatic ring.
  • R 2 is selected from:
  • X is a benzene ring or a 5-6 membered heteroaromatic ring (e.g., a pyridine ring); and Y is absent;
  • R 23 at each occurrence is independently selected from H, halogen, -SF 5 , C 1-6 alkyl, haloC 1-6 alkyl, C 3-6 cycloalkyl, -S( ⁇ O) 2 -(C 1-6 alkyl), -S( ⁇ O) 2 -(C 3-6 cycloalkyl), -P( ⁇ O)(C 1-6 alkyl) 2 , (-C 3-6 cycloalkylene)-CN and (-C 3-6 cycloalkylene)-C 1-6 haloalkyl.
  • R 2 is selected from:
  • R 3 is H or C 1-6 alkyl.
  • R 3 is ethyl
  • Ring Z is a 3-10 membered heterocycle or a benzene ring; preferably a 5-10 membered heterocycle; more preferably a 5-6 membered heterocycle; and
  • heterocyclic ring and the benzene ring are each optionally substituted at each occurrence with one or more substituents independently selected from the group consisting of halogen and C 1-6 alkyl.
  • Ring Z is
  • R 41 is selected from a 3-10 membered heterocycle, a C 6-10 aromatic ring and a 5-14 membered heteroaromatic ring, and the heterocycle, aromatic ring and heteroaromatic ring are each optionally substituted by one or more substituents independently selected from the following: halogen, -OH, -CN, C 1-6 alkyl, -OC 1-6 alkyl and -SC 1-6 alkyl, preferably, the heterocycle, aromatic ring and heteroaromatic ring are at least substituted by -OH, -CN or -OC 1-6 alkyl.
  • R 41 is selected from a 3-10 membered heterocycle, a C 6-10 aromatic ring and a 5-14 membered heteroaromatic ring, and the heterocycle, aromatic ring and heteroaromatic ring are each optionally substituted by one or more substituents independently selected from the following: halogen, -OH, C 1-6 alkyl, -OC 1-6 alkyl and -SC 1-6 alkyl, preferably, the heterocycle, aromatic ring and heteroaromatic ring are at least substituted by -OH or -OC 1-6 alkyl.
  • R 41 is a 5-6 membered heteroaromatic ring (preferably a pyrazole ring or a pyrimidine ring), which is substituted by at least one -OH.
  • R 41 is a 5-membered heteroaromatic ring (preferably a pyrazole ring), which is substituted by at least one -OH.
  • the present disclosure provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, atropisomer, tautomer, polymorph, solvate, metabolite, isotopically labeled compound or prodrug thereof, wherein the compound is selected from:
  • the present disclosure provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, atropisomer, tautomer, polymorph, solvate, metabolite, isotopically labeled compound, or prodrug thereof, wherein the compound is in a non-zwitterionic form, a zwitterionic form, or a mixture of zwitterionic and non-zwitterionic forms;
  • the compound is in any one of the following forms or a mixture of any two or three of them:
  • compositions and methods of treatment are provided.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a preventive or therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, metabolite, isotope-labeled compound or prodrug thereof and one or more pharmaceutically acceptable carriers.
  • the pharmaceutical composition is preferably a solid preparation, a semisolid preparation, a liquid preparation or a gaseous preparation.
  • the pharmaceutical composition may also include one or more other therapeutic agents.
  • the present invention provides the use of a compound of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, metabolite, isotopically labeled compound or prodrug thereof, or a pharmaceutical composition of the present invention in the preparation of a medicament for use as a WRN inhibitor.
  • the present invention provides a compound of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, metabolite, isotopically labeled compound or prodrug thereof, or a pharmaceutical composition of the present invention, for use as a WRN inhibitor.
  • the present invention provides a method for preventing or treating cancer, preferably a cancer characterized by microsatellite high instability (MSI-H) or mismatch repair deficiency (dMMR), comprising administering to an individual in need thereof an effective amount of a compound of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, metabolite, isotopically labeled compound or prodrug thereof, or a pharmaceutical composition of the present invention.
  • MSI-H microsatellite high instability
  • dMMR mismatch repair deficiency
  • the cancer includes colorectal cancer, gastric cancer, endometrial cancer, uterine cancer, adrenocortical cancer, cervical cancer, esophageal cancer, breast cancer, kidney cancer, prostate cancer, and ovarian cancer.
  • “individual” includes humans or non-human animals.
  • Exemplary human individuals include human individuals (referred to as patients) suffering from diseases (e.g., diseases described herein) or normal individuals.
  • “Non-human animals” in the present invention include all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock and/or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).
  • P is a protecting group, preferably an amino protecting group, such as Boc;
  • Lev is independently a leaving group at each occurrence, such as halogen or OH;
  • M is a metal, a borate ester or a boric acid
  • the synthesis route is described by taking the compound with unsubstituted piperazine ring as Z ring as an example.
  • the above general synthesis route can also be used for the synthesis of compounds in which Z ring is substituted piperazine or fused piperazine analogs.
  • A1-1 300 mg, 0.7 mmol
  • A1-2 185 mg, 0.84 mmol
  • 1,4-dioxane 7 mL
  • water (1 mL) and potassium carbonate 210 mg, 1.54 mmol
  • [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (II) 51 mg, 0.07 mmol
  • the reaction solution was returned to room temperature, concentrated to dryness under reduced pressure, and extracted twice with water and dichloromethane.
  • the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain the product A1-3 (90 mg, yield: 24.5%).
  • ESI (m/z) 523.3 [M + H] + .
  • A1-7 (9.8 mg, 0.064 mmol), acetonitrile (1 mL), N-hydroxy-7-azabenzotriazole (8.7 mg, 0.064 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (15 mg, 0.08 mmol) were added to a 25 mL single-mouth bottle in sequence, and stirred at room temperature for 1 hour.
  • A1-6 35 mg, 0.053 mmol
  • N,N-diisopropylethylamine 27 mg, 0.21 mmol
  • A1-1 (650 mg, 1.52 mmol), 4-formylphenylboronic acid pinacol ester (460 mg, 1.98 mmol), 1,4-dioxane (13 mL), water (2 mL) and potassium phosphate (810 mg, 3.8 mmol) were added to a 100 mL single-mouth bottle in sequence.
  • Methanesulfonic acid (2-dicyclohexylphosphino-2',4',6'-tri-isopropyl-1,1'-biphenyl) (2'-amino-1,1'-biphenyl-2-yl) palladium (II) (130 mg, 0.15 mmol) was added under nitrogen protection, and stirred at 100 ° C for 16 hours under nitrogen protection.
  • the reaction solution was returned to room temperature, concentrated to dryness under reduced pressure, and extracted twice with water and dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain the product A2-1 (170 mg, yield: 25%).
  • ESI (m/z) 453.2 [M + H] + .
  • A2-2 (65 mg, 0.12 mmol), N,N-dimethylformamide (1 mL) and N,N-diisopropylethylamine (47 mg, 0.36 mmol) were added to a 25 mL single-necked bottle in sequence, and A1-4 (40 mg, 0.13 mmol) was added in two batches, and stirred at room temperature for 2 hours.
  • Dichloromethane and water were separated and extracted, and the aqueous phase was extracted twice with dichloromethane. The organic phases were combined and washed twice with saturated brine.
  • A1-7 (11 mg, 0.07 mmol), acetonitrile (1 mL), N-hydroxy-7-azabenzotriazole (9.5 mg, 0.07 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (17 mg, 0.087 mmol) were added to a 25 mL single-mouth bottle in sequence, and stirred at room temperature for 1 hour.
  • A2-4 39 mg, 0.058 mmol
  • N,N-diisopropylethylamine (30 mg, 0.23 mmol) were added and stirred at room temperature for 1 hour.
  • A5-1 50 mg, 0.088 mmol
  • DMF 1 mL
  • HOBT 20 mg, 0.15 mmol
  • EDCI 40 mg, 0.21 mmol
  • A4-3 50 mg, 0.088 mmol
  • DIPEA 50 mg, 0.39 mmol
  • acetonitrile 1 mL
  • A4-3 50 mg, 0.088 mmol
  • A6-1 (20 mg, 0.13 mmol)
  • HATU 50 mg, 0.13 mmol
  • DIPEA 50 mg, 0.39 mmol
  • DMF 1 mL
  • A36-1 100 mg, 0.15 mmol
  • A26-1 55 mg, 0.17 mmol
  • potassium carbonate 62 mg, 0.45 mmol
  • 1,4-dioxane 3 mL
  • water 0.4 mL
  • 1,1'-bis(diphenylphosphino)ferrocenepalladium dichloride II was added and stirred at 100 ° C for 16 hours.
  • LCMS showed that the reaction was complete. The materials were fed twice in parallel and combined for post-processing.
  • A85-1 (30 mg, 0.22 mmol), N-hydroxy-7-azabenzotriazole (37 mg, 0.27 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (62 mg, 0.32 mmol) were dissolved in N,N-dimethylformamide (0.5 mL) solution and reacted for 1 hour under stirring at 25°C.
  • Compound A4-3 100 mg, 0.18 mmol
  • N,N-diisopropylethylamine 70 mg, 0.54 mmol
  • A36-1 150 mg, 0.23 mmol
  • potassium carbonate 95 mg, 0.69 mmol
  • 2-methyl-2H-indazole-5-boronic acid 45 mg, 0.25 mmol
  • 1,4-dioxane 1 mL
  • water 0.1 mL
  • 1,1-bis(diphenylphosphino)ferrocenepalladium dichloride II (17 mg, 0.03 mmol) was added and stirred at 80°C for 16 hours.
  • LCMS showed that the reaction was complete, and the reaction solution was poured into water and extracted with dichloromethane (10 mL*2).
  • reaction was quenched with a 10% mass fraction citric acid aqueous solution, extracted twice with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and column chromatography was performed to obtain A20-2 (3.5 g, 33%).
  • A1-7 (1 g, 6.49 mmol), HOBT (0.88 g, 6.43 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.54 g, 8.04 mmol) were dissolved in N, N-dimethylformamide (2 mL) solution and reacted for 1 hour under stirring at 25°C.
  • Compound A20-8 (3 g, 5.36 mmol) and N, N-diisopropylethylamine (2.08 g, 16.08 mmol) were then added to the reaction solution in sequence, and the reaction solution was continued to react for 1 hour under stirring at 25°C.
  • A182-1 (8 g, 49.64 mmol), acetic acid (80 mL) and A182-2 (9.69 g, 74.46 mmol) were added to a 250 mL single-mouth bottle and stirred at 90 ° C for 16 hours.
  • the reaction solution was returned to room temperature, filtered, and the solid was concentrated to dryness to obtain the product A182-3 (10 g, yield: 83.5%).
  • ESI (m/z) 242.2 [M + H] + .
  • A182-3 (10 g, 41.45 mmol), acetonitrile (80 mL) and N,N-diisopropylethylamine (16.1 g, 124.35 mmol) were added to a 250 mL single-mouth bottle, and A20-5 (14.3 g, 43.52 mmol) was slowly added under stirring at room temperature, and stirred at 45 ° C for 16 hours.
  • the reaction solution was returned to room temperature, filtered, and the solid was concentrated to dryness to obtain the product A182-4 (16.4 g, yield: 81%).
  • ESI (m/z) 489.2 [M + H] + .
  • A182-2 (5 g, 34.68 mmol), dichloromethane (50 mL) and sulfonyl chloride (4.91 g, 36.41 mmol) were added to a 100 mL single-mouth bottle and stirred at 25 °C for 16 hours.
  • the reaction solution was extracted three times with water and DCM, and the organic phase was dried over anhydrous sodium sulfate and concentrated to obtain the product 187-1 (6 g, yield: 96.86%).
  • A187-3 (2.87 g, 8.13 mmol), tetrahydrofuran (30 mL), di-tert-butyl dicarbonate (2.66 g, 12.20 mmol) and N,N-diisopropylethylamine (3.15 g, 24.39 mmol) were added to a 100 mL single-mouth bottle and stirred at room temperature for 15 hours.
  • the reaction solution was concentrated, extracted, and subjected to silica gel column chromatography to obtain the product A187-4 (2.4 g, yield: 65.16%).
  • ESI (m/z) 452.3 [MH] - .
  • A187-4 (2.04 g, 4.5 mmol), acetonitrile (20 mL), A20-5 (1.48 g, 4.5 mmol) and N,N-diisopropylethylamine (1.74 g, 13.5 mmol) were added to a 100 mL single-mouth bottle and stirred at 45 ° C for 7 hours.
  • the reaction solution was concentrated and the product A187-5 (0.23 g, yield: 63.41%) was obtained by silica gel column chromatography.
  • ESI (m/z) 699.3 [MH] - .
  • the commercial ADP-Glo assay kit (Promega, #V9102) was used to detect the ADP content generated by the hydrolysis of ATP by WRN helicase, which can reflect the ATPase activity of WRN helicase.
  • nt oligo DNA single strand FLAP26 (TTTTTTTTTTTTTTTTTTTTTTTTTTCCAAGTAAAACGACGGCCAGTGC) was synthesized by Ascent Inc. See, for example, Brosh RM Jr et al., J Biol Chem, 2002 Jun; 277(26):23236-45.
  • reaction buffer (30mM Tris pH 7.5, 2mM MgCl 2 , 0.02% BSA, 50mM NaCl, 0.1% pluronic F127), add 5 ⁇ L 3 ⁇ test compound (diluted to 0.5% DMSO in reaction buffer, final starting concentration is 10 ⁇ M, 1:3 dilution, 9 gradients), 5 ⁇ L 3 ⁇ WRN recombinant protein and 3 ⁇ ATP substrate solution (diluted in reaction buffer, final concentrations of WRN and ATP are 10nM and 300 ⁇ M, respectively) to a 384-well transparent plate, shake and mix, and incubate at 37°C for 3 hours.
  • 5 ⁇ L 3 ⁇ test compound diluted to 0.5% DMSO in reaction buffer, final starting concentration is 10 ⁇ M, 1:3 dilution, 9 gradients
  • 5 ⁇ L 3 ⁇ WRN recombinant protein and 3 ⁇ ATP substrate solution diluted in reaction buffer, final concentrations of WRN and ATP are 10nM and 300 ⁇ M, respectively
  • the compounds of the present application have good inhibitory activity on the ATPase activity of the WRN protein.
  • the WRN gene was stably knocked out in DLD1 cells using CRISPR/Cas9 technology to construct the DLD1-WRN-KO cell line, which was used to evaluate the potential off-target effects of the compounds.
  • Human colon adenocarcinoma cell SW48 cells were cultured in vitro in monolayers, and the culture conditions were: DMEM medium containing 10% fetal bovine serum, 1% penicillin and streptomycin, 37°C 5% CO 2.
  • Human colon adenocarcinoma cell HCT116 cells were cultured in vitro in monolayers, and the culture conditions were: McCoy's5A medium containing 10% fetal bovine serum, 1% penicillin and streptomycin, 37°C 5% CO 2.
  • DLD1-WRN-KO cells were cultured in vitro in monolayers, and the culture conditions were: 1640 medium containing 10% fetal bovine serum, 1% penicillin and streptomycin, 37°C 5% CO 2.
  • SW48 cell line, HCT116 cell line and DLD1-WRN-KO cell line were inoculated in 96-well cell culture plates at an appropriate cell density.
  • the test compound was used to treat the cells with a maximum concentration of 10 ⁇ M, 1:3 times dilution of 9 gradients, and a DMSO treatment group was set up.
  • the cells were balanced at room temperature for 30 minutes, and then 100 ⁇ L of cell proliferation detection reagent CellCounting-Lite (CCL) was added to each well, and incubated in the dark for 10 minutes after oscillation for 5 minutes.
  • CCL cell proliferation detection reagent CellCounting-Lite
  • the chemiluminescence value was read using the Thermo Varioskan LUX-3020 multifunctional microplate reader to convert it into a proliferation index to calculate the inhibition rate of tumor cell proliferation by the compound, and the inhibition rate value and the logarithm of the compound concentration were fitted using nonlinear regression (dose response-variable slope) to obtain the IC 50 value of the compound.
  • the compounds of the present application have good proliferation inhibition activity on microsatellite unstable SW48 cells and HCT116 cells, but have no significant proliferation inhibition activity on WRN knockout DLD1 cells, and have good selectivity.
  • the first-phase metabolic stability of the test compounds was assessed in liver microsomes of CD-1 mice, Sprague-Dawley rats, beagle dogs, cynomolgus monkeys and humans.
  • the animal and human liver microsomes used in this test system were purchased from Xenotech, Corning or other qualified suppliers and stored in a freezer below -60°C before use.
  • test sample and the control compound were incubated with animal and human liver microsomes at 37 ⁇ 1°C for a certain period of time, with the longest incubation time being 60 minutes. Samples were taken out at the specified time point and the reaction was terminated with acetonitrile or other organic solvents containing internal standards. After centrifugation, the resulting supernatant was detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
  • test sample powder is prepared into a stock solution of a certain concentration using DMSO or other organic solvents, and then further diluted with a suitable organic solvent.
  • control compounds testosterone, diclofenac and propafenone were prepared as 10 mM stock solutions in DMSO and then further diluted in appropriate organic solvents.
  • NADP nicotinamide adenine dinucleotide phosphate
  • ISO isocitrate
  • the stop solution is prepared with acetonitrile or other organic solvents containing an internal standard (tolbutamide or other suitable compounds).
  • the prepared stop solution is stored in a refrigerator at 2-8°C.
  • Incubations will be done in 96-well plates. Prepare 8 incubation plates, named T0, T5, T15, T30, T45, T60, Blank60, and NCF60. The first 6 plates correspond to reaction time points of 0, 5, 15, 30, 45, and 60 minutes, respectively. No test or control compound is added to the Blank60 plate, and samples are taken after 60 minutes of incubation. In the NCF60 plate, potassium phosphate buffer is used instead of NADPH regeneration system solution for incubation for 60 minutes. All condition samples are three parallels.
  • the reaction temperature is 37 ⁇ 1°C
  • the final reaction volume is 200 ⁇ L
  • the reaction system includes 0.5mg/mL microsomes, 1.0 ⁇ M substrate, 1mM NADP, 6mM ISO and 1unit/mL IDH.
  • the CV of the internal standard peak area in each matrix should be within 20% for each analytical run.
  • the in vitro elimination rate constant ke of the compound was obtained by converting the ratio of the peak area of the compound to the internal standard into the residual rate in the following formula:
  • CL int(mic) 0.693/T 1/2 /microsomal protein content (microsomal concentration during incubation mg/mL)
  • CL int(liver) CL int(mic) ⁇ amount of microsomal protein in liver (mg/g) ⁇ liver weight to body weight ratio
  • the hepatic intrinsic clearance and hepatic clearance can be converted by the following formula.
  • This experiment was used to test the metabolic stability of compounds in hepatocytes.
  • the samples were mixed at 15, 30, 60, and 90 minutes of incubation, and 25 ⁇ L of the sample was added to 125 ⁇ L of stop solution (containing 200 ng/mL tolbutamide and 200 ng/mL labetalol in acetonitrile) in an ice bath, mixed, and shaken at 500 rpm for 10 minutes. Subsequently, centrifuged at 3220 ⁇ g for 20 minutes at 4 °C. 80 ⁇ L of supernatant was taken from each well and transferred to another 96-well plate containing 240 ⁇ L of ultrapure water. The intrinsic clearance (CLint) and half-life (T1/2) were then analyzed and calculated using LC-MS/MS.
  • stop solution containing 200 ng/mL tolbutamide and 200 ng/mL labetalol in acetonitrile
  • Plasma concentrations were determined using LC-MS/MS.
  • WinNonlin Version 6.3 (Pharsight, Mountain View, CA) pharmacokinetic software was used to process the plasma drug concentration data of the compound using a non-compartmental model.
  • the linear-log trapezoidal method was used to calculate the relevant pharmacokinetic parameters.
  • mice On the day of administration, the actual body weight of the mice was weighed and the administration volume was calculated. There were 9 mice in each group, and two groups of tests were performed for each compound, one group was administered with a single intravenous injection, and the other group of mice was administered with a single oral gavage.
  • Whole blood samples were collected at the specified time (0.25, 0.5, 1, 2, 4, 8, 24h after administration) by orbital bleeding. After blood sample collection, it was immediately transferred to a labeled commercial sample tube containing K2-EDTA (0.85-1.15mg), followed by centrifugation (3200x g, 4°C, 10 minutes) and plasma was collected. The plasma was transferred to a pre-cooled centrifuge tube, snap-frozen in dry ice, and then stored in an ultra-low temperature freezer at -60°C or lower until LC-MS/MS analysis.
  • Plasma concentrations were determined using LC-MS/MS.
  • WinNonlin Version 6.3 (Pharsight, Mountain View, CA) pharmacokinetic software was used to process the plasma drug concentration data of the compound using a non-compartmental model.
  • the linear-log trapezoidal method was used to calculate the relevant pharmacokinetic parameters.
  • Comparative compounds A/C/D are compounds 73/42/125 in patent application WO2022249060, respectively, which are prepared by the synthesis method reported therein, among which comparative compound C is compound HRO761 developed by Novartis and has entered clinical development.
  • Comparative compound B was prepared by referring to the synthesis method of compound A20, except that A1-4 was used instead of A20-5.
  • HEK293 cells were cultured in DMEM medium containing 10% fetal bovine serum and 0.8mg/mL G418 at 37°C and 5% CO2 .
  • the cells were digested with TrypLE TM Express and centrifuged to adjust the cell density to 2 ⁇ 10 6 cells/mL.
  • the cells were then gently mixed on a room temperature balanced shaker for 15-20min and then put on the machine for patch clamp detection.
  • the culture medium of the prepared cells was replaced with extracellular fluid.
  • the intracellular and extracellular fluids were drawn from the liquid pool and added to the intracellular fluid pool, cell and test substance pool of the QPlate chip respectively.
  • the whole-cell patch clamp recorded the voltage stimulation of the whole-cell hERG potassium current, and the experimental data were collected and stored by Qpatch.
  • the compound started at 30 ⁇ M, diluted 3 times, and 6 concentration points were set. Each drug concentration was set to be administered twice for at least 5 minutes. The current detected in the external solution without the compound for each cell was used as its own control group. At least two cells were used for each concentration and the detection was repeated twice independently. All electrophysiological experiments were performed at room temperature.
  • 100mM K-Buffer Mix 9.5mL stock solution A with 40.5mL stock solution B and adjust the total volume to 500mL with ultrapure water. mL, titrate the buffer to pH 7.4 with KOH or H 3 PO 4 .
  • Raw material A (1M potassium dihydrogen phosphate): 136.5 g potassium dihydrogen phosphate in 1 L water;
  • test substance powder is prepared into a stock solution of a certain concentration using DMSO or other organic solvents, and then further diluted with a suitable organic solvent.
  • the in vitro incubation system of liver microsomes for the study of CYP450 enzyme metabolic phenotype is a biochemical reaction carried out under conditions simulating physiological temperature and physiological environment, with the prepared liver microsomes supplemented with redox coenzymes and enzyme-specific selective inhibitors.
  • the concentration of parent drug or its metabolites in the incubation solution was determined by LC-MS/MS.
  • Human colon adenocarcinoma cell SW48 cells were cultured in monolayer in vitro, and the culture conditions were: DMEM medium containing 10% fetal bovine serum, 1% penicillin and streptomycin, and cultured at 37°C 5% CO 2.
  • Human colon adenocarcinoma cell HCT116 cells were cultured in monolayer in vitro, and the culture conditions were: McCoy's5A medium containing 10% fetal bovine serum, 1% penicillin and streptomycin, and cultured at 37°C 5% CO 2. Digestion and passage were performed with trypsin twice a week. When the cell saturation was 80%-90%, the cells were collected, counted, and inoculated.
  • 0.1mL (10 7 ) SW48 cells or 0.1mL (5*10 6 ) HCT116 cells were subcutaneously inoculated into the right back of each mouse.
  • the average tumor volume reached about 150-200mm 3 , and the mice were randomly divided into groups for drug administration, and the drug was administered orally once a day, and the changes in body weight and tumor volume were recorded. After a certain number of days of drug administration, the experiment was terminated. The changes in tumor volume were counted and analyzed. The tumor growth inhibition rate (TGI%) and p value were calculated based on the data on the last day, see the table below.
  • Tumor volume (mm 3 ) 1/2 ⁇ (a ⁇ b 2 ) (Note: a represents the major diameter, b represents the minor diameter)
  • Tumor growth inhibition rate [1-(average tumor volume at the end of a certain medication group - average tumor volume at the beginning of medication in the medication group) / (average tumor volume at the end of treatment in the blank control group - average tumor volume at the beginning of treatment in the blank control group)] ⁇ 100%
  • the p value was calculated based on the relative tumor volume of each mouse in different groups. The p value was obtained by comparing the blank control group and each drug-treated group using the One-way ANOVA method. p>0.05 statistical analysis indicates that there is no significant difference between each group and the blank control group, p ⁇ 0.05 statistical analysis indicates that there is a significant difference between the drug-treated group and the blank control group, and p ⁇ 0.01 statistical analysis indicates that there is a very significant difference between the drug-treated group and the blank control.
  • the p value was obtained by comparing the control compound administration group and the present compound administration group using the One-way ANOVA method. Statistical analysis of p>0.05 indicates that there is no significant difference between the present compound administration group and the control compound administration group. Statistical analysis of p ⁇ 0.05 indicates that there is a significant difference between the present compound administration group and the control compound administration group. Statistical analysis of p ⁇ 0.01 indicates that there is a very significant difference between the present compound administration group and the control compound administration group.

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Abstract

Sont divulgués des composés de formule (I) utiles en tant qu'inhibiteurs de l'hélicase Werner (WRN), une composition pharmaceutique les comprenant, et leur utilisation dans la prévention ou le traitement de cancers caractérisés par une instabilité des microsatellites-élevée (MSI-H) ou une réparation de mésappariements défectueux (dMMR). Drawing_references_to_be_translated :
PCT/CN2024/098964 2023-06-15 2024-06-13 Composés cycliques fusionnés, composition pharmaceutique le comprenant et leur utilisation Pending WO2024255790A1 (fr)

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US12344609B2 (en) 2023-06-08 2025-07-01 Nimbus Wadjet, Inc. WRN inhibitors
WO2025152932A1 (fr) * 2024-01-15 2025-07-24 上海齐鲁制药研究中心有限公司 Dérivé de triazolopyrimidine, son procédé de préparation et son utilisation
WO2025162253A1 (fr) * 2024-02-02 2025-08-07 中国科学院上海药物研究所 Composé spiro, composition pharmaceutique le contenant et son utilisation
WO2025215527A2 (fr) 2024-04-10 2025-10-16 Novartis Ag Combinaisons pharmaceutiques et leurs utilisations

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CN118459466A (zh) * 2023-01-10 2024-08-09 西藏海思科制药有限公司 一种三唑并嘧啶酮衍生物及其在医药上的应用

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WO2024105553A1 (fr) * 2022-11-16 2024-05-23 Novartis Ag Hétérocycles bicycliques et leur utilisation en tant qu'inhibiteurs de wrn
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CN118459466A (zh) * 2023-01-10 2024-08-09 西藏海思科制药有限公司 一种三唑并嘧啶酮衍生物及其在医药上的应用
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WO2024155884A1 (fr) * 2023-01-20 2024-07-25 Synnovation Therapeutics, Inc. Composés hétérocycliques en tant qu'inhibiteurs de wrn
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12344609B2 (en) 2023-06-08 2025-07-01 Nimbus Wadjet, Inc. WRN inhibitors
US12421233B2 (en) 2023-06-08 2025-09-23 Nimbus Wadjet, Inc. WRN inhibitors
WO2025152932A1 (fr) * 2024-01-15 2025-07-24 上海齐鲁制药研究中心有限公司 Dérivé de triazolopyrimidine, son procédé de préparation et son utilisation
WO2025162253A1 (fr) * 2024-02-02 2025-08-07 中国科学院上海药物研究所 Composé spiro, composition pharmaceutique le contenant et son utilisation
WO2025215527A2 (fr) 2024-04-10 2025-10-16 Novartis Ag Combinaisons pharmaceutiques et leurs utilisations

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